New strategy for bioplastic and exopolysaccharides production: Enrichment of field microbiomes with cyanobacteria.

Seven photosynthethic microbiomes were collected from field environmental samples to test their potential in polyhydroxybutyrate (PHB) and exopolysaccharides (EPS) production, both alternatives to chemical-based polymers. Microscope observations together with microbial sequence analysis revealed the microbiome enrichment in cyanobacteria after culture growth under phosphorus limitation. PHB and EPS production were studied under three culture factors (phototrophy, mixotrophy and heterotrophy) by evaluating and optimizing the effect of three parameters (organic and inorganic carbon and days under light:dark cycles) by Box-Behnken design. Results showed that optimal conditions for both biopolymers synthesis were microbiome-dependent; however, the addition of organic carbon boosted PHB production in all the tested microbiomes, producing up to 14 %dcw PHB with the addition of 1.2 g acetate·L-1 and seven days under light:dark photoperiods. The highest EPS production was 59 mg·L-1 with the addition of 1.2 g acetate·L-1 and four days under light:dark photoperiods. The methodology used is suitable for enriching microbiomes in cyanobacteria, and for testing the best conditions for bioproduct synthesis for further scale up.

Production of phycobiliproteins, bioplastics and lipids by the cyanobacteria Synechocystis sp. treating secondary effluent in a biorefinery approach.

Cyanobacteria have been identified as promising organisms to reuse nutrients from waste effluents and produce valuable compounds such as lipids, polyhydroxyalkanoates (PHAs), and pigments. However, almost all studies on cyanobacterial biorefineries have been performed under lab scale and short cultivation periods. The present study evaluates the cultivation of the cyanobacterium Synechocystis sp. in a pilot scale 30 L semi-continuous photobioreactor fed with secondary effluent for a period of 120 days to produce phycobiliproteins, polyhydroxybutyrate (PHB) and lipids. To this end, the harvested biomass from the semi-continuous photobioreactor was transferred into 5 L vertical column batch photobioreactors to perform PHB and lipid accumulation under nutrient starvation. Three hydraulic retention times (HRT) (6, 8 and 10 days) were tested in the semi-continuous photobioreactor to evaluate its influence on biomass growth and microbial community. A maximum biomass concentration of 1.413 g L-1 and maximum productivity of 173 mg L-1 d-1 was reached under HRT of 8 days. Microscopy analysis revealed a shift from Synechocystis sp. to Leptolyngbya sp. and green algae when HRT of 6 days was used. Continuous, stable production of phycobiliproteins in the semi-continuous photobioreactor was obtained, reaching a maximum content of 7.4%dcw in the biomass. In the batch photobioreactors a PHB content of 4.8%dcw was reached under 7 days of nitrogen and phosphorus starvation, while a lipids content of 44.7%dcw was achieved under 30 days of nitrogen starvation. PHB and lipids production was strongly dependent on the amount of nutrients withdrawn from the grow phase. In the case of lipids, their production was stimulated when there was only phosphorus depletion. While Nitrogen and phosphorus limitation was needed to enhance the PHB production. In conclusion, this study demonstrates the feasibility of cultivating cyanobacteria in treated wastewater to produce bio-based valuable compounds within a circular bioeconomy approach.

Monitoring PHB production in Synechocystis sp. with hyperspectral images.

Microalgae wastewater treatment systems have the potential for producing added-value products. More specifically, cyanobacteria are able to accumulate polyhydroxybutyrates (PHBs), which can be extracted and used for bioplastics production. Nonetheless, PHB production requires proper culture conditions and continue monitoring, challenging the state-of-the-art technologies. The aim of this study was to investigate the application of hyperspectral technologies to monitor cyanobacteria population growth and PHB production. We have established a ground-breaking measurement method able to discern spectral reflectance changes from light emitted to cyanobacteria in different phases. All in all, enabling to distinguish between cyanobacteria growth phase and PHB accumulation phase. Furthermore, first tests of classification algorithms used for machine learning and image recognition technologies had been applied to automatically recognize the different cyanobacteria species from a complex microbial community containing cyanobacteria and microalgae cultivated in pilot-scale photobioreactors (PBRs). We have defined three main indicators for monitoring PHB production: (i) cyanobacteria specific-strain density, (ii) differentiate between growth and PHB-accumulation and (iii) chlorosis progression. The results presented in this study represent an interesting alternative for traditional measurements in cyanobacteria PHB production and its application in pilot-scale PBRs. Although not directly determining the amount of PHB production, they would give insights on the undergoing processes.

Process optimization of the polyhydroxybutyrate production in the cyanobacteria Synechocystis sp. and Synechococcus sp.

The effect of four parameters (acetate, NaCl, inorganic carbon and days in darkness) affecting the polyhydroxybutyrate (PHB) production were tested and optimized for Synechococcus sp. and Synechocystis sp. using a Box-Behnken design. The optimal conditions for Synechocystis sp. were found to be 1.2 g L-1 of acetate, 4 gC L-1 of NaHCO3, 18 g L-1 of NaCl and 0 days in darkness. For Synechococcus sp., equal acetate concentration and days in darkness, and lower inorganic carbon and NaCl concentrations than those for Synechocystis sp. were needed (0.05 g L-1 inorganic carbon and 9 g L-1 NaCl). Optimal conditions were scaled up to 3 L photobioreactors. Using Synechocystis sp., 5.6 %dcw of PHB was obtained whether adding or not acetate. In opposition, a maximum of 26.1 %dcw by using acetate was reached with Synechococcus sp. These results provide an easy method to optimize the cultivation conditions to enhance PHB production with cyanobacteria.

Inorganic carbon stimulates the metabolic routes related to the polyhdroxybutyrate production in a Synechocystis sp. strain (cyanobacteria) isolated from wastewater.

Cyanobacteria are capable of transforming CO2 into polyhydroxybutyrate (PHB). In this study, different inorganic carbon concentrations (0-2 gC L-1) were evaluated for a Synechocystis sp. strain isolated from wastewater. Quantitative RT-qPCR was also performed to decipher the links between inorganic carbon and PHB and glycogen metabolism. 2 gC L-1 of bicarbonate stimulated cell growth, nutrients consumption and production of PHB. Using this concentration, a 14%dcw of PHB and an average productivity of 2.45 mgPHB L-1 d-1 were obtained. Gene expression analysis revelated that these conditions caused the overexpression of genes related to glycogen and PHB synthesis. Moreover, a positive correlation between the genes codifying for the glycogen phosphorylase, the acetyl-CoA reductase and the poly(3-hydroxyalkanoate) polymerase was found, meaning that PHB synthesis and glycogen catabolism are strongly related. These results provide an exhaustive evaluation of the effect of carbon on the PHB production and cyanobacterial metabolism.

Optimization of the phototrophic Cyanobacteria polyhydroxybutyrate (PHB) production by kinetic model simulation.

Cyanobacteria can grow using inorganic substrates, such as CO2 from industrial sources and nutrients from wastewaters, and therefore are promising microorganisms to produce polyhydroxybutyrate in a cleaner circular context. However, this biotechnological production is highly challenging because it involves different interlinked reactions that are affected by environmental conditions, which hinders process optimization. In this study a new biokinetic mechanistic model using novel experimental approaches was developed to optimize polyhydroxybutyrate (PHB) and glycogen production. The model includes, for the first time, the production of glycogen and its conversion into PHB, which has been found as the main pathway to produce PHB. Model was successfully (r2: 0.6-0.99) calibrated and validated with experimental data from photobioreactors inoculated with Synechocystis sp. The developed model was used to determine suitable initial conditions for a lab scale batch reactor (6.4 mgN·L-1 and 2 mgP·L-1) and a new configuration for the continuous industrial production of PHB was proposed and optimized using this tool. The maximum productivity (5.1 mgPHB·L-1·d-1) and the optimal configuration and operation of the serial reactors to produce PHB in an industrial scale was achieved using a hydraulic retention time of 4 days in the growth reactor. Then, this reactor daily fed 20 batch accumulation reactors, which were discharged after 20 days. The optimal influent nutrients concentrations for this configuration was found to be 50 mgN·L-1 and 10 mgP·L-1. Results found in this study show the necessity to optimize biopolymers production with Cyanobacteria considering environmental conditions, and demonstrated the potential of this model as a tool to increase PHB productivity.

Free ammonia inhibition in microalgae and cyanobacteria grown in wastewaters: Photo-respirometric evaluation and modelling.

The inhibitory effects of free ammonia (FA) on microalgae/cyanobacteria in wastewater-treating photobioreactors (PBR) can strongly reduce their treatment efficiency, increasing the operational costs and undermining the stability of the system. Although FA-promoting conditions (high pH, temperature and ammoniacal nitrogen concentration) are commonly met in outdoor PBRs, photosynthesis inhibition from FA has been scarcely explored and is rarely considered in microalgae-bacteria growth models. Two pilot systems and a series of lab-scale monocultures were tested using a photo-respirometry approach, to evaluate the effects of FA (8.5-136 mg NH3 L-1) on photosynthesis. Two mathematical inhibition models were compared, with the aim of selecting best-fitting equations to describe photo-respirometric experiments. A set of calibrated inhibition parameters was obtained for microalgae and cyanobacteria, growing in monocultures or in mixed algae-bacteria consortia. Cyanobacteria were more sensitive to FA than green microalgae and mixed phototrophs-bacteria consortia showed a higher resistance compared to monocultures. Estimated inhibition parameters were used to describe common operational/environmental conditions in algae-bacteria systems, demonstrating the potential drop in photosynthetic activity under those relevant operational conditions.

Polyhydroxybutyrate and glycogen production in photobioreactors inoculated with wastewater borne cyanobacteria monocultures.

The aim of this study was to investigate the PHB and glycogen accumulation dynamics in two photobioreactors inoculated with different monocultures of wastewater-borne cyanobacteria, using a three-stage feeding strategy (growth phase, feast-famine phase and feast phase). Two cyanobacterial monocultures containing members of Synechocystis sp. or Synechococcus sp. were collected from treated wastewater and inoculated in lab-scale photobioreactors to evaluate the PHB and glycogen accumulation. A third photobioreactor with a complex microbial community grown in real wastewater was also set up. During each experimental phase different concentrations of inorganic carbon were applied to the cultures, these shifts allowed to discern the accumulation mechanism of carbon storage polymers (PHB and glycogen) in cyanobacteria. Conversion of one into the other was directly related to the carbon content. The highest PHB and glycogen contents (5.04%dcw and 69%dcw, respectively) were achieved for Synechocystis sp.

Band Gap Tuning of Solution-Processed Ferroelectric Perovskite BiFe1-x Co x O3 Thin Films.

Ferroelectric perovskite oxides are emerging as a promising photoactive layer for photovoltaic applications because of their very high stability and their alternative ferroelectricity-related mechanism for solar energy conversion that could lead to extraordinarily high efficiencies. One of the biggest challenges so far is to reduce their band gap toward the visible region while simultaneously retaining ferroelectricity. To address these two issues, herein an elemental composition engineering of BiFeO3 is performed by substituting Fe by Co cations, as a means to tune the characteristics of the transition metal-oxygen bond. We demonstrate by solution processing the formation of epitaxial, pure phase, and stable BiFe1-x Co x O3 thin films for x ≤ 0.3 and film thickness up to 100 nm. Importantly, the band gap can be tuned from 2.7 to 2.3 eV upon cobalt substitution while simultaneously enhancing ferroelectricity. As a proof of concept, nonoptimized vertical devices have been fabricated and, reassuringly, the electrical photoresponse in the visible region of the Co-substituted phase is improved with respect to the unsubstituted oxide.

Selection of cyanobacteria over green algae in a photo-sequencing batch bioreactor fed with wastewater.

In this work, a strategy based on photo-sequencing batch operation was used to select cyanobacteria over unsettled green algae in a wastewater treatment system, evaluating for the first time the effect of hydraulic regimes on nutritional dynamics and microorganisms' competition. During 30 days of operation, an initial microalgae mixed consortia dominated by the green microalgae Scenedesmus sp. was cultivated in two different photo-sequencing batch reactors operated at hydraulic retention time (HRT) of 6 days (PSBR6) and 4 days (PSBR4) at a theoretical solids retention time (SRT) of 10 d. Both reactors were compared with a semi-continuous reactor (SC10) operated at 10 d of HRT and 10 days of SRT (used as a control). The results indicated that PSBR6 and PSBR4 decreased Scenedesmus sp. population by 88% and 48%, respectively. However, only PSBR6 provided suitable conditions to select cyanobacteria from an initial green algae dominated culture. These conditions included volumetric loads of 11.72 mg TN L-1 d-1, 2.04 mg TP L-1 d-1 and 53.31 mg TOC L-1 d-1. The remaining nutrients in the culture led also to a phosphorus limiting N:P ratio (34:1) that improved the increase of cyanobacteria from an initial 2% until 70% of the total population. In addition, PSBR6 reached a biomass production of 0.12 g L-1 d-1, while removing TN, TP and TOC by 58%, 83% and 85%, respectively. Conversely, the application of higher nutrients loads caused by lower HRT (PSBR4) led to an increase of only 13% of cyanobacteria while SC10 remained with the same biomass composition during all the experimental time. Thus, this study showed that the dominance of cyanobacteria in microalgal-based wastewater treatment systems can be controlled by the operational and nutritional conditions. This knowledge could contribute to control microalgae contamination from up-scaling cyanobacterial biomass production in wastewater treatment systems.